Hi Jose, No problem. Thanks for pointing out the issue with comparing different transcripts. Hari On Thu, Jun 9, 2011 at 11:53 AM, <j.delasheras@ed.ac.uk> wrote: > > Hi Hari, > > then I misunderstood what you were trying to do, and Limma will be a great > tool indeed, whether as a two-channel or one-channel mode :) > > Jose > > > > Quoting Hari Easwaran <hariharan.pe@gmail.com> on Thu, 9 Jun 2011 11:33:55 > -0400: > > Hi Jim and Jose, >> Thanks a lot. I will check the limma user guide. >> >> Jose, currently I want to compare expression of transcript-A between >> samples. I see your point why levels of transcript-A and B should not be >> compared. >> >> Sincerely, >> Hari >> >> >> On Thu, Jun 9, 2011 at 7:59 AM, <j.delasheras@ed.ac.uk> wrote: >> >> Quoting Hari Easwaran <hariharan.pe@gmail.com> on Wed, 8 Jun 2011 >>> 12:42:12 >>> -0400: >>> >>> Dear BioC gurus, >>> >>>> >>>> Two-color arrays are generally used to compare expression of a test >>>> sample >>>> (like drug treated) relative to control. Usually Cy5 channel is used for >>>> the >>>> test and Cy3 is used for the control. The output from such data that is >>>> used >>>> for any analysis is the log ratios that gives an idea of relative >>>> expression >>>> of genes in the test vs control samples. >>>> >>>> Can the intensities from an individual channel (Cy3) in a two- color >>>> microarray expression data (in my case Agilent) be used to get a measure >>>> of: >>>> >>>> (a) absolute (approximate) expression level (say E) of a gene/probe >>>> >>>> (b) can the Es for a gene/probe from two or more control samples be >>>> compared >>>> to get an idea of the general expression of that gene in the samples. If >>>> yes, what is the best way to normalize between the Cy3 channels from >>>> different arrays? >>>> >>>> >>>> I guess my question is can the individual channels from a two- color >>>> array >>>> be >>>> used like a one-color array. >>>> >>>> >>>> Sorry if such an issue has been addressed before, but I couldn't get >>>> anything on the web (the keywords to search on such a topic is damn >>>> vague). >>>> >>>> >>>> I would appreciate any help/suggestions. >>>> >>>> >>>> Sincerely, >>>> >>>> Hari >>>> >>>> >>> >>> Dear Hari, >>> >>> there is a way to treat each of a two-colour hyb (no particular >>> convention >>> as to which one is Cy3 and which one is Cy5, in fact many still include >>> dye >>> swap experiments) as a single hyb for various analysis. The Limma user's >>> guide has a chapter on it and it's a good place to start. >>> >>> However, if what you want is to compare relative expression levels >>> between >>> probes/genes to establish that transcript A is expressed 2x the level of >>> transcript B, I don't think you can do that. >>> >>> The reason is that different probes seem to have a different background >>> level and a different intensity range. This is illustrated very clearly >>> if >>> you check the individual probes in a probeset that match a single >>> transcript. Sometimes they all are roughly at the same level, but very >>> often >>> you get some probes that are very low or very high. Very often. >>> For that reason, it is safe to compare the behaviour of the same probe in >>> a >>> range of conditions/samples, but not to compare between probes. >>> You could compare between probes, I suppose, if you use an intermediate >>> comparison, some kind of "normaliser". But I suspect it's not a trivial >>> task >>> to get any kind of trustworthy results. >>> >>> Microarray analyses are best used to compare (many) individual >>> probes/probesets across various conditions or samples, whether using a >>> single channel or a two channel approach, it doesn't really change >>> matters, >>> and even that is not very quantitative unless you include and use >>> standards >>> in your array design & hybridisation. >>> >>> Jose >>> >>> >>> -- >>> Dr. Jose I. de las Heras Email: >>> J.delasHeras@ed.ac.uk >>> The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131 6507090 >>> Institute for Cell & Molecular Biology Fax: +44 (0)131 6507360 >>> Swann Building, Mayfield Road >>> University of Edinburgh >>> Edinburgh EH9 3JR >>> UK >>> >>> >>> -- >>> The University of Edinburgh is a charitable body, registered in >>> Scotland, with registration number SC005336. >>> >>> >>> >>> >> > > > -- > Dr. Jose I. de las Heras Email: J.delasHeras@ed.ac.uk > The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131 6507090 > Institute for Cell & Molecular Biology Fax: +44 (0)131 6507360 > Swann Building, Mayfield Road > University of Edinburgh > Edinburgh EH9 3JR > UK > > > -- > The University of Edinburgh is a charitable body, registered in > Scotland, with registration number SC005336. > > > [[alternative HTML version deleted]]